Page 69 - Haematologica March 2020

Genetic and epigenetic regulation of latexin transcription
and determine the effect of SNP rs31528793 on HMGB2 binding in vivo, we performed ChIP-qPCR assay on BM cells of C57BL/6 and DBA/2 mice which naturally carry the SNP. The binding affinity of HMGB2 was quantita- tively measured by real-time PCR with primers spanning the promoter sequence containing G/C SNP (see also Figure 1C). We did not detect any difference in the binding affinity, suggesting that the G/C allelic variant does not cause differential binding of HMGB2 to Lxn promoter (data not shown). Altogether, this evidence strongly sug- gests that HMGB2 and SNP rs31528793 act independently to regulate Lxn transcription.
HMGB2 is a member of the high mobility group family proteins. It is a non-histone chromatin-binding protein that remodels chromatin architecture, therefore affecting gene expression. HMGB2 has been shown to play an important role in maintaining stem cell population in a tis- sue-specific manner. For example, in the nervous system, HMGB2 deletion leads to the increased neural stem/prog- enitor cells by increasing their proliferation.41 However, in articular cartilage, loss of HMGB2 reduces the regenera- tive capacity of mesenchymal stem cells by increasing apoptosis.42 Similarly, knockdown of HMGB2 decreased the number of muscle stem (satellite) cells by inhibiting proliferation and stimulating differentiation, thereby lead- ing to the impaired muscle regeneration.43 Our study showed that the functional effects of HMGB2 on HSC and the blood system are similar to those in mesenchymal and muscle stem cells. Knockdown of HMGB2 decreased HSC number and blood regeneration by increasing apoptosis and decreasing proliferation. These effects are mediated, at least in part, via the upregulation of Lxn, which is a neg- ative regulator of HSC function. HMGB2 has also been shown to play an important role in cellular senescence and aging.44,45 It binds to the chromosome loci of key senescence-associated secretory phenotype (SASP) genes and prevents their incorporation into transcriptionally repressive heterochromatin environment during senes- cence, thereby inducing SASP gene expression. Since we found that Lxn is one of the transcriptional targets of HMGB2, whether Lxn is involved in senescence and aging remains a subject of great interest and remains to be determined. Our unpublished data show that Lxn expres- sion increases with aging, and old HSC with Lxn deple- tion have the increased regenerative capacity that is com- parable to young HSC. Thus, inhibition of Lxn may reju- venate old HSC.
Natural genetic variation is associated with a variety of
hematologic phenotypes in humans. Genome-wide asso- ciation studies have revealed DNA variants that are impli- cated in hematologic traits such as fetal hemoglobin levels, hematocrit, cell counts and sizes of different types of blood cells, as well as in disease susceptibility.10 One of the best examples of the functional effect of genetic varia- tion is a regulatory SNP that causes the blood disorder α-thalassemia. This SNP creates a new transcriptional pro- moter that interferes with normal transcription of α-glo- bin genes and leads to disease development.11 However, very few genes underlying the vast majority of these DNA variants have been uncovered and very little is known about how they contribute to the phenotypic diversity in the population.9 Lxn is the first stem cell regulatory gene reported that accounts for the natural diversity of HSC function.17 Here, also for the first time, we discovered that SNP rs31528793 is one of the DNA variants that are asso- ciated with the differential expression of Lxn in mouse. The Lxn gene is evolutionarily conserved. Since it is iden- tified by the genetic diversity that arises through natural selection, it may physiologically regulate a function in other natural populations, such as humans. In fact, our preliminary data have indicated that there is also a nega- tive correlation between Lxn level and the number of HSC and HPC in healthy humans (C Zhang et al., 2019, unpub- lished data). Therefore, Lxn may be involved in human hematopoiesis and there might be polymorphisms in human genome that are functionally similar to mouse SNP rs31528793. Interestingly, a recent report has shown that a SNP rs6441224 in Lxn promoter is associated with its expression level in humans.46 So it would be very interest- ing to determine whether HMGB2 binds to this SNP-con- taining promoter region and regulates human Lxn and HSC function. These would become very useful genetic markers for screening of transplantation donors with a larger stem cell reservoir or for prediction of better recov- ery of cancer patients from the therapy-induced BM and stem cell suppression.
Acknowledgments
The authors are supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under awards R01HL124015(YL), R21HL140213 (YL), UKY-CCTS UL1TR001998 (UKY-CCTS pilot), and the Markey Cancer Center’s Biostatistics and Bioinformatics Shared Resource Facility as well as the Flow Cytometry Shared Resource Facility (P30CA177558). We thank the Markey Cancer Center's Research Communications Office for editing and graphics support.
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